Chondrocyte proliferation and differentiation requires their attachment to the collagen type II–rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (α-NAC). Based on sequence similarities between α-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of α-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434–440, 1994), we investigated a putative role for α-NAC/1.9.2 in transcriptional control. The α-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because α-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of α-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between α-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the α-NAC/1.9.2 gene product functions as a transcriptional coactivator.

The treatment of choice for pseudo-vitamin D deficiency rickets (PDDR), caused by mutations in the 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1; 1α-OHase) gene, is replacement therapy with 1,25(OH)2D3. We have previously engineered an animal model of PDDR by targeted inactivation of the 1α-OHase gene in mice. Replacement therapy was performed in this model. The 1α-OHase−/− mice and heterozygote controls were treated with 500 pg of 1,25(OH)2D3/g body weight/day for 2 weeks, followed by 100 pg of 1,25(OH)2D3/g body weight/day for an additional 3 weeks before death at 8 weeks of age. Blood biochemistry analysis revealed that the rescue treatment corrected the hypocalcemia and secondary hyperparathyroidism. The daily injections of 1,25(OH)2D3 induced strong expression of CYP24, the 25-hydroxyvitamin D 24-hydroxylase gene. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue regimen also restored the biomechanical properties of the bone tissue within normal parameters. These results show that chronic treatment with the active 1,25(OH)2D3 metabolite is effective to rescue the PDDR phenotype of 1α-OHase mutant mice.

The biological activity of 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃] remains controversial, but it has been suggested that it contributes to fracture healing. Cyp24a1^–/– mice, synthesizing no 24R,25(OH)₂D₃, show suboptimal endochondral ossification during fracture repair, with smaller callus and reduced stiffness. These defects were corrected by 24R,25(OH)₂D₃ treatment, but not by 1,25-dihydroxyvitamin D₃. Microarrays with Cyp24a1^–/– callus mRNA identified FAM57B2 as a mediator of the 24R,25(OH)₂D₃ effect. FAM57B2 produced lactosylceramide (LacCer) upon specific binding of 24R,25(OH)₂D₃. Fam57b inactivation in chondrocytes (Col2-Cre Fam57b^fl/fl) phenocopied the callus formation defect of Cyp24a1^–/– mice. LacCer or 24R,25(OH)₂D₃ injections restored callus volume, stiffness, and mineralized cartilage area in Cyp24a1-null mice, but only LacCer rescued Col2-Cre Fam57b^fl/fl mice. Gene expression in callus tissue suggested that the 24R,25(OH)₂D₃/FAM57B2 cascade affects cartilage maturation. We describe a previously unrecognized pathway influencing endochondral ossification during bone repair through LacCer production upon binding of 24R,25(OH)₂D₃ to FAM57B2. Our results identify potential new approaches to ameliorate fracture healing.